Human gene mutations causing infertility.

The identification of gene mutations causing infertility in humans remains noticeably deficient at present. Although most males and females with infertility display normal pubertal development, nearly all of the gene mutations in humans have been characterised in people with deficient puberty and subsequent infertility. Gene mutations are arbitrarily categorised into four different compartments (I, hypothalamic; II, pituitary; III, gonadal; and IV, outflow tract). Diagnoses of infertility include hypogonadotrophic hypogonadism (compartments I and II), hypergonadotrophic hypogonadism (III), and obstructive disorders (compartment IV). Most gene mutations identified to date affect gonadal function, but it is also apparent that a large number of important genes in normal fertility have yet to be realised.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

Mutations of follicle stimulating hormone-beta and its receptor in human and mouse: genotype/phenotype.

The pituitary gonadotropin follicle stimulating hormone (FSH) interacts with its membrane-bound receptor, to produce biologic effects. Traditional functions of FSH include, follicular development and estradiol production in females and the regulation of Sertoli cell action and spermatogenesis in males. FSHbeta knock-out mice and transgenic mice, serve as models for FSH deficiency and excess, respectively. In addition, mutations of both FSHbeta and FSHR genes have been characterized in humans, although phenotypic effects of the ligand appear to be more profound than those of its receptor. FSH is essential for normal puberty and fertility in females, particularly ovarian follicular development beyond the antral stage. In males, FSH is necessary for normal spermatogenesis and when FSH function is completely absent, infertility occurs. With partial FSH deficiency in males, spermatogenesis is affected, but fertility may still be possible. FSH may also be necessary for normal androgen synthesis in males and females.

Genetics of human hypogonadotropic hypogonadism.

Humans with hypogonadotropic hypogonadism (HH) manifest irreversible pubertal delay, infertility, and low serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Although the genetic basis of this condition is largely unknown, mutations have been identified in approximately 5-10% of HH patients. Mutations in the KAL gene (Kallmann syndrome) and the AHC gene (adrenal hypoplasia congenita/HH) cause X-linked recessive HH. Autosomal recessive HH may be brought about by mutations in the gonadotropin-releasing hormone receptor, leptin, and the leptin receptor genes. Isolated deficiencies of the gonadotropins FSH and LH are due to corresponding beta-subunit genes. PROP1 gene mutations lead to combined pituitary deficiency, and HESX gene mutations result in septo-optic dysplasia, both of which include HH. These identified gene mutations advance our understanding of normal hypothalamic-pituitary-gonadal function.

Mutations in the follicle-stimulating hormone-beta (FSH beta) and FSH receptor genes in mice and humans.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein synthesized in the anterior pituitary gland, is important for the production of sex steroids and gametes. FSH-beta (FSH beta) and FSH receptor (FSHR) knockout mice display impaired ovarian follicular development and infertility in females and small testes, oligospermia, and fertility in males. Humans with FSH beta gene mutations tend to have a more severe phenotype than those with FSHR gene mutations, although infertility and varying degrees of impaired sex steroid production occur in both types of mutations. Data from human and mouse mutations in the FSH beta and FSHR genes suggest that FSH is necessary for normal pubertal development and fertility in males and females.

Mutations in human gonadotropin genes and their physiologic significance in puberty and reproduction.

OBJECTIVE: Human gene mutations provide an opportunity to study the pathophysiology of the disease process as well as normal physiology. The purpose of the present report was to review known human gene mutations that affect gonadotropin secretion. DESIGN: A retrospective analysis of studies of human gene mutations that affect hypothalamic, pituitary, and gonadal function was conducted. RESULT(S): Mutations have been identified for at least three genes that cause inherited hypogonadotropic hypogonadism. In addition, gene mutations for the beta-subunits of FSH and LH have been characterized. Both activating and inactivating mutations have been identified for the gonadotropin receptor genes. CONCLUSION(S): The identification of human gene mutations has furthered our understanding of the normal processes of pubertal development and fertility.

The Finnish follicle-stimulating hormone receptor gene mutation is rare in North American women with 46,XX ovarian failure.

OBJECTIVE: To determine whether FSH receptor gene missense mutation in Finnish women with premature ovarian failure (POF) is present in North American women with POF. DESIGN: Analysis of DNA from patients and controls. PATIENT(S): Thirty-five women with POF and ten normal controls. INTERVENTION(S): Extraction of DNA with subsequent digestion by the enzyme BsmI, polyacrylamide gel electrophoresis, ethidium bromide staining, and photography. MAIN OUTCOME MEASURE(S): After restriction enzyme digestion, the frequencies of the normal allele (two fragments of 51 and 27 base pairs) and the mutant allele (a single 78-base pair fragment) were determined. RESULT(S): BsmI digestion was noted for all 35 affected individuals and 10 controls, thus demonstrating homozygosity for the normal FSH receptor allele. No patient or control was heterozygous or homozygous for the mutant allele. CONCLUSION(S): The missense mutation in the human FSH receptor gene in Finnish women with POF is uncommon in North American women with POF. The molecular basis of ovarian failure for most patients remains unknown.

The detection of Hind III restriction-fragment length polymorphisms using a deoxyribonucleic acid probe for the beta subunit of follicle-stimulating hormone.

Molecular diagnosis of disorders of follicle-stimulating hormone (FSH) production may become possible now that the gene for FSH beta has been characterized. Restriction-fragment length polymorphism (RFLP) analysis provides a means of organized search for molecular variants of FSH. The purpose of this study was to screen controls for the presence of RFLPs using the deoxyribonucleic acid (DNA) probe pFSH beta -1.4. Genomic DNA was digested with 12 different restriction endonucleases; Southern blots were constructed and hybridized to pFSH beta -1.4. No polymorphisms were identified with 11 enzymes. Three of 24 (12.6%) Hind III digests demonstrated a polymorphic fragment of either 5.2, 4.7, or 4.3 kb. These are the first RFLPs identified for the FSH beta gene with pFSH beta -1.4. RFLPs for FSH beta constitute the first step in the molecular analysis of disorders of FSH production.

Follicle-stimulating hormone beta gene structure in premature ovarian failure.

OBJECTIVE: To determine whether mutations in the gene for FSH beta are present, and possibly etiologic, in some patients with 46,XX premature ovarian failure (POF). DESIGN: DNA samples obtained from 18 study patients with POF and two menopausal fertile controls were studied by Southern blot analysis. DNA sequencing was performed in one patient. SETTING: Patients were seen in a reproductive endocrinology clinic and studied in a medical school laboratory setting at the Medical College of Georgia and Tufts University. MAIN OUTCOME MEASURES: Restriction fragment sizes on autoradiographs were compared between the study group and controls. DNA sequencing radiographs were compared between one study patient and five controls. RESULTS: Fragment sizes obtained with the restriction enzymes EcoRI, DraI, HincII, PstI, KpnI, BglI, BamHI, and BglII were similar size in both study subjects and controls using the probes pFSH beta-1.4 and pFSH beta-1.2. A previously described HindIII polymorphism was present using pFSH beta-1.2, but HindIII fragment sizes were identical in patients with ovarian failure and controls using pFSH beta-1.4. DNA sequencing of the FSH beta gene in one patient was normal. CONCLUSIONS: No mutations in the gene for FSH beta were identified in women with POF. DNA sequencing of the exons and promoter region of the FSH beta gene in one woman with POF was normal. This does not entirely exclude the possibility that smaller deletions, insertions, or point mutations of the FSH beta could be etiologic in some women with POF. The HindIII polymorphism does not appear to segregate with 46,XX POF.

The follicle-stimulating hormone receptor gene is polymorphic in premature ovarian failure and normal controls.

OBJECTIVE: To examine the FSH receptor gene for detectable abnormalities in women with premature ovarian failure. DESIGN: Study of genomic DNA from controls and from patients with 46,XX premature ovarian failure (POF). SETTING: Clinics and laboratories of university gynecology and obstetrics departments. PATIENTS: Twenty-one women with 46,XX POF and 40 normal fertile controls. INTERVENTIONS: Deoxyribonucleic acid was analyzed in patients and controls by Southern blot analysis, polymerase chain reaction (PCR), and denaturing gradient gel electrophoresis. Southern blots were hybridized with the FSH receptor complementary DNA and other smaller DNA probes. Exons 1, 5 to 6, and 10 were amplified by PCR and electrophoresed on agarose gels. Polymerase chain reaction products from exons 1 and 10 were electrophoresed on denaturing gradient gels. MAIN OUTCOME MEASURES: Fragments obtained by Southern blot analysis and PCR were compared in patients and controls. Polymerase chain reaction fragments electrophoresed on denaturing gels also were compared in patients and controls. CONCLUSIONS: No FSH receptor gene deletions or other mutations were identified in women with POF. Southern blots containing PstI- and HindIII-digested DNA revealed restriction fragment length polymorphisms in both patients and controls. Denaturing gradient gel electrophoresis analysis of PCR fragments of exon 10 also demonstrated DNA sequence polymorphisms in both patients and controls. Follicle-stimulating hormone receptor gene deletions are not common in women with POF, although the gene is polymorphic. We cannot exclude point mutations in other regions of the FSH receptor gene in some patients with POF.

Gonadotropin-releasing hormone-associated peptide gene sequences in women with hyperprolactinemia.

OBJECTIVE: To determine if mutations in the structural gene for gonadotropin-releasing hormone (GnRH)-associated peptide are present in women with hyperprolactinemia. DESIGN: Patients with hyperprolactinemia and controls were studied retrospectively for GnRH-associated peptide gene mutations. SETTINGS: Patients seen in a clinical setting were studied at a medical school laboratory setting. PATIENTS: Fifteen women with hyperprolactinemia and two fertile controls with normal prolactin levels were studied. INTERVENTIONS: Genomic deoxyribonucleic acid (DNA) was extracted from each patient and subjected to Southern blot analysis and polymerase chain reaction (PCR). For Southern blot analysis, DNA was digested with EcoRI, XbaI, BglII, PstI, and BamHI and hybridized to two DNA probes for GnRH-associated peptide. Exons II to IV, which encode for the structural gene, were amplified by PCR. MAIN OUTCOME MEASURES: Fragment sizes from autoradiographs were compared among patients and controls. Amplified PCR products of exons II to IV of the GnRH-associated peptide were also compared. RESULTS: No large deletions, insertions, or polymorphisms were identified in women with hyperprolactinemia or controls by Southern blotting. Each of the exons was present and of normal size by PCR in the study patients and controls. CONCLUSIONS: No large deletions of the GnRH-associated peptide gene appear to be present in our patients with hyperprolactinemia. Small deletions, insertions, or point mutations are not excluded by this analysis.

Identification of restriction-fragment length polymorphisms for the human chorionic gonadotropin-beta/luteinizing hormone-beta gene cluster.

OBJECTIVE: To determine whether restriction fragment length polymorphisms are present using a deoxyribonucleic acid (DNA) probe for human luteinizing hormone beta subunit (hLH-beta). If the gene for hLH-beta is polymorphic, genetic diagnosis of disorders of luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) production could become possible. DESIGN: Study of genomic DNA from controls with a variety of restriction enzymes to identify polymorphisms. SETTING: Laboratories of the Department of Obstetrics and Gynecology, Department of Oral Biology, Medical College of Georgia, Augusta, Georgia. PATIENTS: Unrelated control men and women seen in clinics at the Medical College of Georgia. INTERVENTIONS: Genomic DNA was extracted from patients and digested with eight different restriction enzymes for the study of the hLH-beta gene by Southern analysis. MAIN OUTCOME MEASURE: Fragment (band) sizes on radiographs from Southern blots were compared with those from molecular weight standards. CONCLUSIONS: Restriction fragment length polymorphisms were identified for four of the restriction enzymes, DraI, HincII, MboI, and KpnI. These polymorphisms may be useful in the diagnosis of disorders of hLH and hCG production.

Gonadotropin-releasing hormone, follicle-stimulating hormone beta, luteinizing hormone beta gene structure in idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine if the genes for gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone beta (FSH beta), and luteinizing hormone beta (LH beta) are present, and if so, whether gene structure is normal in patients with idiopathic hypogonadotropic hypogonadism (IHH). DESIGN: Patients with clinical and laboratory characteristics of IHH were studied at the deoxyribonucleic acid (DNA) level to assess gene structure. SETTING: This study took place in an academic setting. PATIENTS: Human volunteers with documented IHH and fertile controls were studied. INTERVENTIONS: Genomic DNAs were extracted from each patient, Southern blots were constructed and hybridized to DNA probes for GnRH, FSH beta, and LH beta. DNA samples were also subjected to polymerase chain reaction analysis. MAIN OUTCOME MEASURES: Gene structure was assessed by analysis of autoradiographs and gel electrophoresis of polymerase chain reaction products in both the study patients and controls. RESULTS: Each analysis for FSH beta, LH beta, and GnRH demonstrated the same sized fragments in both the study group and control group. A 1.2-kilobase fragment containing the coding region for GnRH was present in all patients with IHH and controls by polymerase chain reaction. CONCLUSIONS: The genes for GnRH, LH beta, and FSH beta are present in patients with IHH. No large deletions or rearrangements of any of these genes were identified in any of these patients.

Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.

OBJECTIVE: To determine the extent of paternal and maternal chromatin decondensation in unfertilized eggs after intracytoplasmic sperm injection (ICSI). DESIGN: Eggs that failed to show two pronuclei (2-PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). PATIENT(S): Forty-nine patients undergoing IVF cycles. MAIN OUTCOME MEASURE(S): The unfertilized eggs were studied by chromatin staining. RESULT(S): The average fertilization rate from all ICSI cycles in these two groups was 45%. The fertilization rates in groups A and B were 35% and 59%, respectively. In group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin, whereas 45% were partially decondensed. In these two groups, no sperm chromatin was detected in 24% of the unfertilized eggs. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. CONCLUSION(S): These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause of failure of fertilization rates in ICSI cycles. Rather, it is likely to be the failure to complete both the maternal and paternal chromatin transitions that occur with normal fertilization.

Ultrasound prediction of follicle volume: is the mean diameter reflective?

OBJECTIVE: To evaluate the relationship between 2 dimensional sonographic measurement of ovarian follicles and their actual volume. DESIGN: Prospective clinical study. SETTING: The in vitro fertilization (IVF) program of a University based, tertiary care hospital. PATIENTS AND INTERVENTIONS: Sonographic categorization by shape, and measurement of 96 individual ovarian follicles immediately prior to aspiration for IVF. Each follicle was aspirated under direct ultrasound guidance and the volume recorded. The 96 follicles were visualized in a total of 14 patients from whom 2 to 27 oocytes were obtained. MAIN OUTCOME MEASURE: Total volume of each follicle. RESULTS: Round and polygonal follicles exhibited a highly significant relationship between sonographically measured mean diameter and total follicle volume. The volume of follicles that were categorized as ellipsoid was not predicted by measurement of the longest diameter, shortest diameter or mean diameter. CONCLUSION: The mean diameter of round and polygonal follicles accurately predicts total follicular volume. However, clinical decisions in ovulation induction should be modified when the follicle shape is predominantly ellipsoid because the traditionally held belief that the sonographic measurement of the follicular diameter correlates with the follicular volume does not apply in those circumstances.

Mutation analysis of the gonadotropin-releasing hormone receptor gene in idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine if GnRH receptor mutations occur in patients with idiopathic hypogonadotropic hypogonadism. DESIGN: Patients and controls were studied by molecular genetic analysis. SETTING: A tertiary medical center setting. PATIENT(S): Twenty-four patients with idiopathic hypogonadotropic hypogonadism and 20 controls. INTERVENTION(S): Deoxyribonucleic acid from all individuals was analyzed by Southern blot analysis and denaturing gradient gel electrophoresis. Genomic DNA was digested with restriction enzymes, and Southern blots and denaturing gradient gel blots were constructed. Blots were hybridized with the GnRH receptor complementary DNA probe. The DNA sequencing was performed on samples from two representative patients. MAIN OUTCOME MEASURE(S): Gonadotropin-releasing hormone receptor gene structure was ascertained by comparing fragments from autoradiographs in patients and controls. Individual nucleotides were ascertained from DNA sequencing gels. RESULT(S): No GnRH receptor gene deletions or polymorphisms were identified by Southern blot analysis. New restriction-fragment melting polymorphisms using the enzymes DpnII, RsaI, and HaeIII were identified by denaturing gradient gel blots in patients and controls. CONCLUSION(S): Gonadotropin-releasing hormone receptor gene deletions or rearrangements were not observed in our idiopathic hypogonadotropic hypogonadism patients. Denaturing gradient gel electrophoresis failed to identify single-base differences unique to patients with idiopathic hypogonadotropic hypogonadism, dramatically reducing the likelihood that point mutations of the GnRH receptor gene are present in idiopathic hypogonadotropic hypogonadism.

Molecular analysis of the gonadotropin-releasing hormone receptor in patients with polycystic ovary syndrome.

OBJECTIVE: To determine whether a mutation in the GnRH receptor gene is responsible for polycystic ovary syndrome (PCOS). DESIGN: Molecular analysis of human genomic DNA. SETTING: Academic research environment. PATIENT(S): Eighty patients with PCOS. INTERVENTION(S): Extraction and polymerase chain reaction (PCR) analysis of genomic DNA, confirmation of PCR products by ethidium bromide staining of agarose gels after electrophoresis, denaturing gradient gel electrophoresis of PCR products, and photography. MAIN OUTCOME MEASURE(S): Mutations in the GnRH receptor of women with PCOS. RESULT(S): Denaturing gradient gel electrophoresis revealed no mutations in the exonic sequence encoding the open reading frame of the GnRH receptor. CONCLUSION(S): A mutation in the GnRH receptor gene is unlikely to be the underlying cause of PCOS in most patients. The molecular basis of this disorder remains unknown.

Administration of progesterone before oocyte retrieval negatively affects the implantation rate.

OBJECTIVE: To compare the efficacy of two clinically accepted methods of progesterone supplementation during IVF. DESIGN: Prospective randomized trial. SETTING: A university-based IVF program. PATIENT(S): Three hundred fourteen stimulated IVF cycles between January 1993 and October 1994. INTERVENTION(S): Patients were assigned to one of two luteal phase progesterone regimens by a random permuted block design. In protocol A, 12.5 mg of IM progesterone was given 12 hours before oocyte retrieval; in protocol B, 25 mg of IM progesterone was given on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Patient demographic characteristics, including age, diagnosis, number of oocytes retrieved and fertilized, and number of embryos transferred, were not different between the two groups. There was no difference in the rate of cycle cancellation between the groups. One hundred forty ETs were performed in patients assigned to protocol A and 142 in patients assigned to protocol B. The clinical pregnancy rate in group A was 12.9% compared with 24.6% in group B. CONCLUSION(S): The administration of progesterone before oocyte retrieval is associated with a lower pregnancy rate than the administration of progesterone after oocyte retrieval.

Mutation analysis of the EMX2 gene in Kallmann's syndrome.

OBJECTIVE: To investigate the possibility that a mutation in the human EMX2 gene may be involved in Kallmann's syndrome. DESIGN: In vitro experiment. SETTING: Academic Medical Center. PATIENTS: One hundred and twenty patients with Kallman's syndrome or idiopathic hypogonadotrophic hypogonadism (IHH). INTERVENTION: Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOMES MEASURES: Single-stranded conformational polymorphism (SSCP) analysis was used to identify possible mutations of the EMX2 gene. RESULTS: One hundred and twenty patients with Kallmann's syndrome or IHH, had no mutations noted in this gene. CONCLUSION: It is unlikely that EMX2 mutations are a clinically significant cause of IHH or Kallman's syndrome.